The invention relates to methods for image processing, in particular a method for the reconstruction of images that have been recorded with a microscope using structured illumination, and an imaging method for microscopic imaging of an object. The invention also relates to devices for carrying out said methods, in particular an imaging apparatus, for example a microscope, and a reconstruction device for image reconstruction, and applications of the method.
In optical microscopy, various methods have been developed for imaging objects with resolution in depth, for example, to obtain a section image in a particular region through a sample or, by means of recording a plurality of section images, to obtain a three-dimensional representation of the sample. For example, in confocal microscopy, using confocal, point illumination and imaging, image information is gathered only in the sectional plane of interest. Disadvantages arise in confocal microscopy, in particular, due to the complex microscope configuration and the complex gathering of image information.
Another method is microscopy with structured illumination microscopy (SIM). With this wide field microscopy method, a periodic grating structure is focused onto the sectional plane of interest in the sample. By imaging the grating structure in the sectional plane, a modulation of the illumination is introduced into the coordinate space and thereby the “missing cone” problem of wide field microscopy is overcome. From a plurality of intermediate images, each of which is recorded with a different phase position of the grating structure, an image function can be calculated which represents an image of the sectional plane in the real object. Conventional microscopy with structured illumination, however, has the general disadvantage that the image reconstructed from the intermediate images represents an approximation the quality of which may be impaired, in particular, by artifacts and instabilities.
In WO 2004/038483 A1 and in the publication by L. H. Schaefer et al. (“Journal of Microscopy”, vol. 216, 2004, pp. 165-174), a method for microscopy with structured illumination is described with which the image reconstruction is improved. On the basis of a reconstruction algorithm, which describes the gathering of image information using a three-dimensional model, it was proposed that, for example, brightness variations in the object illumination or occurrences of bleaching in the object that can occur over time during recording of the intermediate images, should be taken into account in the image reconstruction. In practice, however, it has been found that the quality of the image reconstruction suffers from undesirable limitations in special application cases, in particular when investigating biological samples. These are expressed therein that, for example, the details of the real sample are imaged with an insufficient level of resolution.
It is an objective of the invention to provide an improved method for image reconstruction from intermediate images of an object illuminated with structured illumination, and with which the disadvantages of conventional techniques can be overcome. It is intended, in particular, that the reconstruction method should enable an imaging of optical sections with improved spatial resolution. It is a further objective of the invention to provide an improved imaging method for obtaining images of objects with structured illumination. It is a further objective of the invention to provide a correspondingly improved imaging apparatus.
This objective is solved with the method and devices of the invention.